Numerous polypeptide molecules cause an allergic reaction in man and animals. Hay fever, more particularly ragweed sensitivity, characterized classically by itching of the mucosa of the nose, mouth, pharynx and eyes, lacrimation, sneezing, watery nasal discharge, coughing and sometimes asthmatic wheezing afflicts a considerable number of humans. Sensitivity to bee venom is characterized classically by a wheal and flare reaction, swelling, anaphylaxis and occasionally death. Food allergies classically are manifested by urticaria, perhaps nausea, diarrhea, and hives.
It is known that the appearance of allergy or atopy is involved with production of a tissue-sensitizing immunoglobulin (IgE) antibody. These IgE antibodies have an affinity for receptors on cells in various body tissues. The receptors on mast cells and basophils are of special significance since these cells contain pharmacologically-active mediators, such as histamine, serotonin, and kinins concentrated in cytoplasmic granules. When IgE antibodies are fixed to mast cells and basophils, contact with antigens can result in cross-linking. This cross-linking causes degranulation of mast cells and basophils which in turn release the pharmacologically active mediators (particularly histamine) and which are responsible for manifestations of the allergic response.
Thus, these disturbances are caused by immunoglobulin E reacting with specific polypeptide molecules in these substances. Untreated, these disturbances can lead to asthma in the case of hayfever, disabling reactions in the case of insect venom and limited dietary intake in the case of foods. Treatment for hayfever usually entails trial of orally active antihistamines, sometimes in combination with sympathomimettes such as phenylpropanolamine or phenylephrine. The symptoms may thus be partially suppressed, but the basic physiological difficulty, the histamine release by the antibody antigen reaction, persists. Treatment of insect stings by these agents is not practical since it is not possible to know when the event may occur and the reaction can be very severe if treatment is not immediate. Similarly the food reaction must have immediate treatment to achieve its effect. Commonly these treatments are inadequate or unsatisfactory and resort must be had to a more fundamental treatment, particularly for the pollen and venom allergies, namely desensitization.
One of the conventional and widely used methods of treating allergy is by immunizing or desensitizing a patient by repeated injections of small, gradually increasing amount of antigen, which is believed to increase the levels of blocking immunoglobulin G (IgG) antibodies and at the same time to arrest the production of IgE antibodies. This apparently conflicting effect of antigen administration is not fully understood but apparently involves differences in immunoregulation of two different classes of antibodies, viz. IgG and IgE.
This desensitization is undertaken with extreme caution. Commencing with a very dilute preparation of ragweed allergen or bee venom allergen, injected subcutaneously, if there is no local reaction within an hour of injection, the next dose is doubled and administered in 3 or 4 days. To be used in the event of an anaphylactic reaction, there must be available for emergency administration, adrenaline, antihistamines and intravenously administrable antiinflammatory corticosteroids during the testing and desensitization procedures. Both the time and precautions accompanying the conventional ragweed desensitization procedures are inconvenient. Benefits obtained by such treatments are frequently inconsistent.
This invention is concerned with the discovery, manufacture and method of use of a new and specific polypeptide immunosuppressant fraction produced by controlled proteolytic digestion of a polypeptide allergen which, upon administration for the specific allergic reaction in mammals, including humans, effects protection against said allergens without the accompanying and dangerous possibility of anaphylactic shock and other disadvantages indicated above. Pollen allergen preparations of this invention are made, illustratively, from common ragweed, giant ragweed, rye (Groups I, II and III), June grass, orchard grass, sweet vernal grass, red top, timothy, yellow dock, wheat, corn, sagebrush, blue grass, California annual grass, pig weed, Bermuda grass, Russian thistle, mountain cedar, oak, box elder, sycamore, maple, elm, etc. Ragweed constitutes the most prevalent and noxious of these and is the primary example illustrated herein. However, the pollen allergens of grasses and trees are chemically similar to ragweed pollen, the major alergens being acidic proteins of varying molecular weights within the range of about 20,000 to 40,000, as described by T. P. King, Advances In Immunology, 77-105, 1976, Academic Press, New York, N.Y. This publication further characterizes bee venom allergens (principally phospholipase and hyaluronidase) and food allergens (e.g. ovalbumin and ovomucoid) as being similar to the inhalant allergens, all being globular proteins.
According to King antigen E of short ragweed pollen comprises two non-identical polypeptide chains held together in the native molecule by noncovalent forces, the chains having molecular weights of about 26,000 and 13,000 daltons, respectively. Ovalbumin is reported as having a molecular weight of 44,000, while ovamucoid is a glycoprotein having a molecular weight of about 27,000. Bee venom phospholipase has a molecular weight of about 15,800, and hyaluronidase has a molecular weight of about 50,000. Both are basic glycoproteins.
King further reports that ragweed pollen antigen E is stable toward proteolytic digestion at neutral pH by trypsin, chymotrypsin and papain, but is digested by nagarase at neutral pH. Nagarase-digested antigen E showed less than 0.001% of the allergenic activity of intact antigen E. Ryegrass allergens Groups I and II are readily digested by trypsin and chymotrypsin, with complete removal of the antigenic and allergenic activities as well as haptenlike inhibitory activity, in the case of Group I.
The purpose of the proteolytic digestion reported by King was to show that allergens were polypeptides. The products of digestion described by King exhibited entirely different properties from those of the present invention.
As a general observation King states:
"The dominant antigenic determinants of the major allergens of ragweed and ryegrass pollens, codfish, and bee venom are dependent on both the primary structure and the conformation of the molecule, a property in common with the other globular protein antigens."
An article by L. Berrens, Annuals New York Academy of Sciences, 221: 183-198, 1974, attributes allergenic activity to lysine-sugar Amadori products which were present in all the allergens examined by him, based on the following observations: "Mild oxidation of these atopic allergens, using alkaline potassium ferric cyanide, hydrogen peroxide, or ultraviolet radiation, which selectively destroyed the highly vulnerable 1-amino-1-deoxy-2 ketose side chains, caused allergenic activity to diminish, without serious damage to the antigenic integrity of the carrier molecule.
The synthetic introduction of (lysine)-sugar structures of the above configuration into inactive carrier proteins led to the production of allergenically active preparations, featuring all the characteristic chemical properties of `natural` allergens.
In the light of these results, we have gradually come to regard the `lysine-sugar` site as essential in priming the allergic reaction."
Insect venom allergen preparations of this invention are made, illustratively, from bee venom, yellow jacket venom and other insects of the Hymenoptera order.
Food allergen preparations of this invention are made from egg, milk, and other sources of similar allergenic proteins.
The process of the present invention, involving controlled proteolytic enzyme digestion, is operable with all allergens of the above types of which applicants are aware. The polypeptide active immunosuppressant fractions of this invention, in contrast to prior art allergens, can be administered without likelihood of anaphylaxis.
Attempts to achieve separation of protective and allergic inducing activities for the treatment of pollen sensitivities, particularly in ragweed-sensitive individuals, have been reported. For example, Ishizaka, K., et al, J. Immunol. 113: 70-77, 1974, prepared four modification of the active allergenic fraction of ragweed pollen extract, antigen E, namely urea denatured antigen E (UD), its alpha and beta polypeptide chains and a reduced carboxymethylated antigen E (RC). All modifications lost antigenic determinants present in the unmodified antigen E and accordingly could not be used to achieve satisfactory antibody production against antigen E in humans. Each of the four preparations failed to combine with human gamma globulin, human IgG, against the original native antigen, and did not induce erythema wheal reactions in ragweed sensitive individuals. Since modified antigens do not induce allergenic reactions in the patients but are capable of stimulating T cells, carrier-specific helper cells, Ishizaka speculated that such modified antigens may change the T cell population without side effects. Subsequently, Ishizaka, et al., J. Immunol., 114: 110-115, 1975, reported that test results collectively indicate that a major population of B cells stimulated by native antigen is different from the majority of B cells stimulated by the urea-modified antigen. Further work on UD or the alpha-polypeptide chain isolated from the denatured molecule provided additional evidence that these materials prime T cells specific for native antigen E (Takatsu, et al., J Immunol., 115: 1469-1476, 1975 and 116: 1257-1264, 1976). It must be recognized that the denatured protein and polypeptide chains obtained from the denatured protein themselves introduce foreign material which could aggravate allergic sensitivity by providing yet another foreign sensitizing material, over and above ragweed antigen, to which the body may react.
In addition to urea-denatured antigens described by Ishizaka et al, which do not react with antibodies to native antigen molecules, the literature has disclosed other modified antigen preparations. These may be summarized as follows:
Glutaraldehyde polymerized antigen: R. Patterson, J. Allergy and Clinical Immunology, 68: 85-90, 1981 immunogenic, but reactive and administrable only in small amounts. PA0 Formaldehyde treated antigen: D. G. Marsh et al, J. Allergy and Clinical Immunology, 68: 449-459, 1981--non-reactive but ineffective in immunosuppression. PA0 Antigen conjugated with d-amino acids or polyethylene glycol: F. Liu et al, Proc. National Academy of Sciences USA, 76: 1430-1434, 1979; and A. H. Sehon et al, J. Allergy and Clinical Immunology, 64: 242-250, 1979--immunosuppressive, reactivity similar to unmodified antigenic extracts.
The art has thus failed to show or suggest the preparation of a pollen desensitizing agent free of anaphylactic reactivity, such as could be used therapeutically.